Cutting Edge: Foxp1 Controls Naive CD8+ T Cell Quiescence By

Cutting Edge: Foxp1 Controls Naive CD8+ T Cell Quiescence by Simultaneously Repressing Key Pathways in Cellular Metabolism and Cell Cycle Progression This information is current as of September 28, 2021. Hairong Wei, Jianlin Geng, Bi Shi, Zhenghui Liu, Yin-Hu Wang, Anna C. Stevens, Stephanie L. Sprout, Min Yao, Haikun Wang and Hui Hu J Immunol 2016; 196:3537-3541; Prepublished online 21 March 2016; Downloaded from doi: 10.4049/jimmunol.1501896 http://www.jimmunol.org/content/196/9/3537 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2016/03/18/jimmunol.150189 Material 6.DCSupplemental References This article cites 28 articles, 10 of which you can access for free at: http://www.jimmunol.org/content/196/9/3537.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Th eJournal of Cutting Edge Immunology

Cutting Edge: Foxp1 Controls Naive CD8+ T Cell Quiescence by Simultaneously Repressing Key Pathways in Cellular Metabolism and Cell Cycle Progression Hairong Wei,* Jianlin Geng,* Bi Shi,* Zhenghui Liu,* Yin-Hu Wang,* Anna C. Stevens,* Stephanie L. Sprout,* Min Yao,† Haikun Wang,† and Hui Hu* Previously we have shown that transcription factor Previously we have shown that transcription factor Foxp1 Foxp1 plays an essential role in maintaining naive plays a critical role not only in the generation of quiescent T cell quiescence; in the absence of Foxp1, mature naive naive T cells during thymocyte development but also in the + CD8 T cells proliferate in direct response to homeo- maintenance of quiescence of mature naive T cells in the Downloaded from static cytokine IL-7. In this study, we report that the periphery (7, 8). Compared with the other reported T cell deletion of Foxp1 in naive CD8+ T cells leads to en- quiescence genes (3–6), Foxp1 regulation of T cell quiescence shows a unique propensity in that the acute deletion of Foxp1 hanced activation of the PI3K/Akt/mammalian target + of rapamycin signaling pathway and its downstream in mature naive CD8 T cells allows the cells to proliferate in cell growth and metabolism targets in response to IL-7. direct response to the stimulation of homeostatic cytokine IL- We found that Foxp1 directly regulates PI3K interacting 7 (8). The underlying mechanism, however, is still largely http://www.jimmunol.org/ protein 1, a negative regulator of PI3K. Additionally, unknown. When cells proliferate, cell metabolism and growth increase we found that deletion of Foxp1 in naive CD8+ T cells to both maintain the homeostatic control of cell size and results in increased expression levels of E2fs, the crit- support the cell division. In T lymphocytes, the PI3K/Akt/ ical components for cell cycle progression and prolif- mammalian target of rapamycin (mTOR) pathway is crucial eration, in a manner that is not associated with for cell survival, growth, and proliferation (9–11). Recently, increased phosphorylation of retinoblastoma protein. PI3K interacting protein 1 (Pik3ip1) has been identiﬁed as a Taken together, our studies suggest that Foxp1 en- + novel negative regulator that functions upstream of PI3K, by guest on September 28, 2021 forces naive CD8 Tcellquiescencebysimulta- leading to decreased Akt phosphorylation (12, 13). In addi- neously repressing key pathways in both cellular tion to cell growth, cell cycle progression must also occur for metabolism and cell cycle progression. The Journal cells to proliferate. Studies have shown that DNA replication of Immunology, 2016, 196: 3537–3541. and cell cycle progression are controlled by the tumor suppressor retinoblastoma protein (Rb)/E2F pathway (14–17). The sequential phosphorylation of Rb by different cyclin–cdk ymphocyte quiescence refers to a cellular state char- complexes inactivates Rb, releasing E2F factors (E2F1, E2F2, acterized by small cell size, low metabolic activity, and and E2F3) from the repression E2F–Rb complexes (18–21). L lack of spontaneous proliferation. A considerable In T lymphocytes, it has been shown that E2F1 and E2F2 amount of literature has suggested that the quiescence of naive play important roles in maintaining T cell homeostasis; the T cells in the periphery is not a default state: the survival of deletion of both proteins diminishes lymphopenia-induced quiescent naive T cells requires constant subthreshold signals T cell proliferation (22). from the engagement of the TCR and stimulation of IL-7R (1, In this study, we further investigated the underlying 2), and multiple factors, including transcription factors, have mechanism by which Foxp1 regulates naive T cell quiescence. been found to actively maintain T cell quiescence (3–6). We show that Foxp1 deletion in mature naive CD8+ T cells

*Department of Microbiology, School of Medicine, University of Alabama at Birming- Address correspondence and reprint requests to Dr. Haikun Wang or Dr. Hui Hu, Key ham, Birmingham, AL 35205; and †Key Laboratory of Molecular Virology and Immu- Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chi- nology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200031, nese Academy of Sciences, Room B411, Life Science Research Building, 320 Yueyang China Road, Xuhui District, Shanghai 200031, China (H. Wang) or Department of Micro- biology, School of Medicine, University of Alabama at Birmingham, 845 19th Street ORCIDs: 0000-0003-4894-9744 (Z.L.); 0000-0002-5326-4996 (A.C.S.); 0000-0003- South, BBRB Room 859, Birmingham, AL 35205 (H.H.). E-mail addresses: hkwang@ips. 0081-0460 (M.Y.); 0000-0002-4714-4672 (H. Wang). ac.cn (H. Wang) or [email protected] (H.H.). Received for publication August 25, 2015. Accepted for publication February 29, 2016. The online version of this article contains supplemental material. This work was supported by Knowledge Innovation Program Grant Y414P11212 from Abbreviations used in this article: ChIP, chromatin immunoprecipitation; cKO, condi- the Chinese Academy of Sciences and the China Young 1000-Talent Program (to tional knockout; KO, knockout; DKO, double knockout; mTOR, mammalian target of H. Wang), funds from the Alliance for Cell Gene Therapy Foundation (to H.H.), Na- rapamycin; Pik3ip1, PI3K interacting protein 1; Rb, retinoblastoma protein; WT, wild- tional Institutes of Health Grants AI095439 and AI103162 (to H.H.), a University of type. Alabama at Birmingham Center for AIDS Research vaccine concept grant (to H.H.), and by University of Alabama at Birmingham Center for AIDS Research Grant Ó P30AI027767-26. Copyright 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501896 3538 CUTTING EDGE: Foxp1 REPRESSES KEY METABOLISM AND CELL CYCLE PATHWAYS results in enhanced activation of the PI3K/Akt/mTOR path- shown previously (8), naive YFP+ Foxp1f/fCre-ERT2+RosaYFP way, and Foxp1 regulates Pik3ip1 directly. We also found that (Foxp1–conditional knockout [cKO]) CD8+ T cells but not Foxp1 deficiency results in increased expression levels of E2fs naive Foxp1f/fRosaYFP (Foxp1–wild-type [WT]) CD8+ T cells and that E2F1 indeed contributes to the proliferation of proliferated and increased cell size in response to IL-7 in vitro Foxp1-deficient naive CD8+ T cells in response to IL-7. (Fig. 1A). Interestingly, we found that Ly294002 and rapamycin, the inhibitors of PI3K and mTOR, respectively, suf- Materials and Methods ficiently abrogated both the proliferation and the increased Mice cell size of Foxp1-cKO CD8+ T cells in response to IL-7 All mice were maintained in specific pathogen-free barrier facilities and were (Fig. 1A). We further examined the activation of Akt. In used in accordance with protocols approved by the Institutional Animal Care Foxp1-cKO CD8+ T cells cultured with IL-7 for a total of 4 2/2 and Use Committee of the University of Alabama at Birmingham. E2f1 d, a time point at which the cells had not proliferated but a mice were from The Jackson Laboratory. Foxp1ATg/TgCD4-Cre and Foxp1f/f Cre-ERT2+RosaYFP mice were generated as described (8). Foxp1f/fCre-ERT2+ significant fraction of the cells were in the S phase (data not 2 2 2 2 RosaYFP mice were crossed with E2f1 / mice to generate E2f1 / Foxp1f/f shown), the phosphorylation of Akt was markedly enhanced Cre-ERT2+RosaYFP mice. compared with that in control Foxp1-WT CD8+ T cells Flow cytometry and cell sorting (Fig. 1B). Furthermore, the phosphorylation of p70S6 kinase and its substrate ribosomal protein S6 was induced in Flow cytometry and cell sorting were done as described (8). All cell sorting was + done on a FACSAria (BD Biosciences). The sorted populations were .98% Foxp1-cKO CD8 T cells (Fig. 1C). Previously we have pure. Abs were as follows: Alexa Fluor 700–anti-CD44 (IM7, BioLegend) and shown that Foxp1 deletion leads to elevated IL-7R expression Downloaded from Brilliant Violet 785–anti-CD8a (53-6.7, BioLegend). The Live/Dead fixable (8). To determine whether enhanced Akt and p70S6 kinase aqua dead cell fluorescence kit was from Invitrogen. activity in Foxp1-cKO CD8+ T cells is mainly caused by the CellTrace Violet labeling and cell culture elevated IL-7R expression, we cultured both Foxp1-WT and Foxp1-cKO CD8+ T cells with a high dosage of IL-7 (15 ng/ml) A CellTrace Violet cell proliferation kit (Invitrogen) was used for analysis of cell proliferation. CD44loCD8+ T cells sorted from Foxp1f/fRosaYFP, Foxp1f/f that nearly saturated the activation of IL-7R/Akt signaling 2 2 2 2 Cre-ERT2+RosaYFP, E2f1 / Foxp1f/fRosaYFP,andE2f1 / Foxp1f/fCre-ERT2+ http://www.jimmunol.org/ RosaYFP mice were labeled with CellTrace Violet following the manufacturer’s instructions. CellTrace Violet–labeled CD8+ T cells were cultured for 6 d with or without recombinant murine IL-7 (R&D Systems) and 0.3 mM4- hydroxytamoxifen (Sigma-Aldrich). For chemical inhibition experiments, 10 mM Ly294002 (PI3K inhibitor, Calbiochem) and/or 10 nM rapamycin (inhibitor, MBL International) were added to the culture at day 0 and cell proliferation was analyzed at day 6. For enforced Pik3ip1 overexpression, Pik3ip1 was subcloned from pEF-PIK3IP1 (Addgene, no. 49214) into the retroviral vector MSCV-IRES-Thy1.1 and retroviruses were produced as described (23). Cells were transduced with virus-containing medium sup- by guest on September 28, 2021 plemented with Polybrene (6 mg/ml) twice at day 4 by a plate centrifugation method. Akt phosphorylation and cell proliferation were analyzed at day 6. Real-time RT-PCR CD8+ T cells cultured with IL-7 were harvested at indicated time points. RNA was purified as described (8) for real-time PCR analysis of Pik3ip1, E2f1, E2f2, E2f3, Cdk1, Mcm5, and Pcna mRNAs. The primers are provided in Supplemental Table I. Immunoblot analysis CD8+ T cells cultured with IL-7 for a total of 4 d were harvested for immunoblot analysis. Cells were lysed and SDS-PAGE was done as described (8). Abs to phospho-S6 Rb (2F9), phosho-p70 S6 kinase (Ser371), Rb (D20), phospho-Rb (C84F6), and phospho-Akt (9271) were from Cell Signaling Technology. b-Actin (I-9) was from Santa Cruz Biotechnology. Pik3ip1 Ab (16826-1-AP) was from Proteintech. Chromatin immunoprecipitation Chromatin immunoprecipitation (ChIP) assay was done as described (8). Foxp1-precipitated DNA and input DNA were assessed by quantitative real- time PCR with universal SYBR Green supermix (Bio-Rad).

Statistical analysis FIGURE 1. Foxp1 deletion in naive CD8+ T cells leads to enhanced ac- A two-tailed Student t test was used when two groups were compared for tivation of PI3K/AKT/mTOR pathway in response to IL-7. (A) Naive CD8+ statistical differences. An ANOVA test was used when more than two groups T cells from Foxp1f/fRosaYFP or Foxp1f/fCre-ERT2+RosaYFP mice were labeled were compared for statistical differences. with CellTrace Violet and cultured with tamoxifen and IL-7 (3 ng/ml) in the absence or presence of 10 mM Ly294002 and/or 10 nM rapamycin. At day 6, Results and Discussion the proliferation and the size of the cultured cells were examined. (B and C) Foxp1 deﬁciency in naive CD8+ T cells leads to enhanced activation of Naive CD8+ T cells from Foxp1f/fRosaYFP or Foxp1f/fCre-ERT2+RosaYFP mice the PI3K/Akt/mTOR pathway in response to IL-7 were cultured with tamoxifen and IL-7 (3 ng/ml) for 2 d. Foxp1-WT or Foxp1-cKO (YFP+) T cells were then sorted and cultured with IL-7 (3 ng/ml) To determine whether the PI3K/Akt/mTOR pathway plays for another 2 d; at day 4, the cultured cells were harvested for immunoblot a role in Foxp1-mediated quiescence regulation, we ﬁrst used analysis of p-Akt (B), p70S6K, and S6 (C). b-Actin served as a loading the pharmacological inhibitor blocking approach. As we have control. Data in (A)–(C) are representative of three independent experiments. The Journal of Immunology 3539

(Supplemental Fig. 1A). We found that the phosphorylation escence by negatively regulating key pathways in cellular of p70S6 kinase and S6 was induced only in Foxp1-cKO metabolism and cell growth. + CD8 T cells (Supplemental Fig. 1B), suggesting that the + + Foxp1 deficiency in naive CD8 T cells leads to induction of proliferation of Foxp1-deficient CD8 T cells in response to E2F factors IL-7 is not simply due to the elevated IL-7R; rather, there are also other Foxp1 targets involved in promoting the cell To determine how Foxp1 deficiency affects cell cycle pro- proliferation. gression, we first examined the phosphorylation of Rb. Rb has Taken together, these results suggest that Foxp1 deletion in multiple phosphorylation sites, and studies have shown that + the Rb phosphorylated at S780 cannot bind to E2F1 in vivo naive CD8 T cells results in increased PI3K/Akt/mTOR + signaling in response to IL-7 and this pathway plays an (18, 21). In naive CD8 T cells that were cultured with IL-7 for a total of 4 d, we found that the phosphorylation of Rb at important role in Foxp1-mediated regulation of T cell + quiescence. S780 remained at basal levels in Foxp1-cKO CD8 T cells as in control Foxp1-WT CD8+ T cells (Fig. 3A). We also did Foxp1 directly regulates the expression of Pik3ip1 not find any differences at some other phosphorylation sites of Rb between Foxp1-WT and Foxp1-cKO CD8+ T cells Studies have shown that Pik3ip1 is a negative regulator of PI3K (Supplemental Fig. 1C). Surprisingly, however, by day 4, (13). Interestingly, we found that in the cultures with IL-7, + the expression of E2f1, E2f2,andE2f3 mRNAs in Foxp1- Foxp1-cKO CD8 T cells expressed significantly lower +

+ cKO CD8 T cells was induced to significantly higher levels Downloaded from levels of Pik3ip1 protein compared with WT CD8 T cells + + than in Foxp1-WT CD8 T cells at the IL-7 concentrations (Fig. 2A). Consistently, we found that Foxp1-cKO CD8 adequate enough to induce cell proliferation (Figs. 3B, 4A). T cells expressed significantly lower levels of Pik3ip1 mRNA, Consistently, the expression levels of E2F targets Cdk1 (24, whereas Foxp1A transgene enhanced its expression (Fig. 2B), 25), Mcm5 (26), and Pcna (24, 25), which are all crucial for suggesting that Pik3ip1 could be a direct target of Foxp1. We cell cycle progression, were also induced to higher levels in performed the bioinformatics analysis and identified one +

Foxp1-cKO CD8 T cells (Fig. 3C). The expression of E2fs http://www.jimmunol.org/ forkhead binding site with high scores in the promoter region and their target genes was also examined at early time points of the Pik3ip1 locus (Fig. 2C, left panel). The ChIP assay of of the culture before the cells entered the cell cycle. We found + Foxp1 in mature WT CD8 T cells showed that Foxp1 that the mRNAs of E2f1, E2f3, Cdk1, Mcm5, and Pcna, but bound speciﬁcally to the Pik3ip1 promoter region (Fig. 2C, not E2f2, were already induced to higher levels in Foxp1-cKO right panel). To further address the function of Pik3ip1, we than in Foxp1-WT CD8+ T cells (Supplemental Fig. 1D, used retroviral expression approach and found that the over- 1E). The results suggest that in response to IL-7, the in- + expression of Pik3ip1 in Foxp1-cKO CD8 T cells reduced creased expression levels of cell cycle–related genes in Foxp1- the Akt phosphorylation levels and the cell proliferation in deleted CD8+ T cells are directly due to Foxp1 deﬁciency and response to IL-7 (Fig. 2D). As expected, the overexpression of are not associated with increased phosphorylation of Rb. In- by guest on September 28, 2021 Foxp1A in Foxp1-cKO CD8+ T cells also reduced the Akt terestingly, note that at a relatively low IL-7 concentration of phosphorylation levels and the cell proliferation in response to 0.6 ng/ml, Cdk1 mRNA levels were increased in Foxp1-cKO IL-7 (Fig. 2D). These results suggest that Foxp1 likely CD8+ T cells in the absence of the induction of E2fs (Fig. dampens PI3K/Akt/mTOR signaling via its direct control of 3C). The increased expression levels of E2f1–3 at two higher Pik3ip1 expression levels. Thus, Foxp1 enforces T cell qui- IL-7 concentrations helped boost the Cdk1 levels even higher

FIGURE 2. Foxp1 directly regulates the expression of Pik3ip1. (A) Naive CD8+ T cells from Foxp1f/fRosaYFP or Foxp1f/fCre-ERT2+RosaYFP mice were cultured with tamoxifen and IL-7 (3 ng/ml) for 2 d, and Foxp1-WT and Foxp1-cKO T cells were then sorted and cultured with IL-7 (3 ng/ml) for another 2 d before being harvested for immunoblot analysis of Pik3ip1 protein. (B) Naive CD8+ T cells from Foxp1f/fRosaYFP, Foxp1f/fCre-ERT2+RosaYFP,orFoxp1ATg/TgCD4-Cre mice were cultured with tamoxifen and IL-7 (3 ng/ml) for 2 d, and Foxp1-WT, Foxp1-cKO, and Foxp1A transgenic (Foxp1A Tg) T cells were then sorted and cultured with IL-7 (3 ng/ml) for another 2 d before being harvested for real-time PCR analysis of Pik3ip1 mRNA; results are normalized to Rpl32 mRNA and are presented relative to the Pik3ip1 mRNA in Foxp1-WT CD8+ T cells. (C) Predicted forkhead binding site (underlined) in the Pik3ip1 promoter (left panel) and ChIP analysis of the binding of Foxp1 to a control region (Ctrl) or the promoter region (Prom) of Pik3ip1, presented as binding in Foxp1-WT CD8+ T cells relative to binding in Foxp1-cKO CD8+ T cells (right panel). (D) Naive CD8+ T cells from Foxp1f/fCre-ERT2+RosaYFP mice were labeled with CellTrace Violet and cultured with tamoxifen and IL-7 (3 ng/ml). Cells were transduced with retroviruses at day 4; Akt phosphorylation and cell proliferation were analyzed by ﬂow cytometry at day 6. Numbers indicate the mean ﬂuorescence intensity of staining. Data in (B) and (C) are shown as mean 6 SEM of three independent experiments. Data in (A) and (D) are representative of two independent experiments. **p , 0.01, ***p , 0.001. 3540 CUTTING EDGE: Foxp1 REPRESSES KEY METABOLISM AND CELL CYCLE PATHWAYS Downloaded from http://www.jimmunol.org/

FIGURE 4. E2F1 contributes to the cell proliferation of Foxp1-deficient naive CD8+ T cells in response to IL-7. Naive CD8+ T cells from Foxp1f/f 2 2 2 2 RosaYFP, Foxp1f/fCre-ERT2+RosaYFP, E2f1 / Foxp1f/fRosaYFP,orE2f1 / by guest on September 28, 2021 Foxp1f/fCre-ERT2+RosaYFP mice were (A) labeled with CellTrace Violet and FIGURE 3. Foxp1 deletion leads to increased levels of E2f1-3. Naive CD8+ cultured with tamoxifen and different concentrations of IL-7 for 6 d before B T cells from Foxp1f/fRosaYFP or Foxp1f/fCre-ERT2+RosaYFP mice were cul- the cultured cells were analyzed for cell proliferation, or ( ) cells were cultured with tamoxifen and different concentrations of IL-7 for 2 d; Foxp1-WT tured with tamoxifen and IL-7 (3 ng/ml) for 2 d, and Foxp1-WT, Foxp1- or Foxp1-cKO T cells were then sorted and cultured with IL-7 (at the same cKO, E2F1 KO, or E2F1/Foxp1 DKO T cells were then sorted and cultured concentrations as before) for another 2 d. At day 4, the cultured cells were with IL-7 (3 ng/ml) for another 2 d. At day 4, the cultured cells were har- harvested for (A) immunoblot analysis of total Rb and phosphorylated Rb, vested for real-time PCR analysis of E2f1, E2f2, and E2f3 mRNAs; results are and (B and C) real-time PCR analysis of (B) E2f1, E2f2, and E2f3 mRNAs, normalized to Rpl32 mRNA and are presented relative to the corresponding + A and (C) cdk1, mcm5, and pcna mRNAs in Foxp1-WT and Foxp1-cKO CD8+ mRNAs in Foxp1-WT CD8 T cells. Data in ( ) are representative of three B 6 T cells; results are normalized to Rpl32 mRNA and are presented relative to independent experiments; data in ( ) are shown as mean SEM of three , , the corresponding mRNAs in control Foxp1-WT CD8+ T cells. Data in (A) independent experiments. *p 0.05, **p 0.01. are representative of three independent experiments; data in (B) and (C) are shown as mean 6 SEM of three independent experiments.**p , 0.01, ***p , + 0.001. eration of Foxp1-deficient CD8 T cells in response to IL-7. However, the deletion of E2f1 in Foxp1-cKO CD8+ T cells did not completely abrogate cell proliferation (Fig. 4A). We in Foxp1-cKO CD8+ T cells (Fig. 3C). These results suggest found that in the absence of E2f1, the expression levels of that Foxp1 regulates important pathways involved in cell cycle E2f2 and E2f3 were still induced to higher levels in Foxp1- progression. cKO CD8+ T cells than in Foxp1-WT CD8+ T cells (Fig. 4B), E2F1 plays an important role in regulating T lymphocyte suggesting that the increased levels of E2f2 and E2f3 likely homeostasis (22). Early studies have shown that E2F1 stim- compensate for the E2f1 deficiency and contribute to the re- ulates its own transcription and both E2f2 and E2f3 are direct sidual cell proliferation of DKO CD8+ T cells in response to targets of E2F1 (27, 28). To determine whether the increased IL-7. Taken together, our results suggest that via its repression levels of E2fs contribute to the cell proliferation of Foxp1- on the expression levels of E2F factors, Foxp1 enforces T cell deficient CD8+ T cells in response to IL-7, we generated quiescence by negatively regulating cell cycle progression. 2 2 E2f1 / Foxp1f/fCre-ERT2+RosaYFP mice. We found that In summary, we have shown that Foxp1 functions as a Foxp1-cKO CD8+ T cells proliferated extensively in response critical regulator of T cell quiescence by simultaneously to IL-7 by day 6 in vitro, whereas naive CD8+ T cells deficient repressing key pathways in both metabolism and cell cycle of both E2f1 and Foxp1 (double KO [DKO]) proliferated progression. Foxp1 directly regulates a negative regulator of much less (Fig. 4A), suggesting that increased E2f1 expression PI3K, Pik3ip1, the overexpression of which reduces both the levels in Foxp1-cKO CD8+ T cells contribute to the prolif- Akt phosphorylation and the cell proliferation in response to The Journal of Immunology 3541

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